Motor unit number estimation in normal and parkinsonism model of medial gastrocnemius muscle in rats.

Motor units (MUs) reflect the function of the central nervous motor system. Thus, the estimated MU number is a good option to investigate the functional movement disorder in the Parkinson's disease (PD). The purpose of this study was to compare the estimated MUs number in the medial gastrocnemius (MG) muscle of the normal rats and those with the parkinsonism. The MG muscle of two age-matched group (normal and parkinsonism) of anesthetized male, adult (154-304 days old) Wistar rats were studied after the insertion of electromyography (EMG) needles. The insertion activity and the MU recruitment (MUR), the strengths of mechanical involuntary contractions and the evoked spike potentials, were recorded. The means initial and the maximal amplitudes of the motor unit potentials (MUPs) were calculated for the estimated MUs number. The spinal cord at the L4-L6 was removed for pathological study. The parkinsonism MUPs trace showed irregular and low threshold discharge rate. The normal spikes trace, however, was different. Increased age was not associated with any increase in the MU number in the two groups. However, there was a significant correlation between the mentioned parameters and the insertion activity (r= -0.25, r= -0.177) and the MUR (r= 0.86, r= 0.248) in the normal and the parkinsonism groups, respectively. There was a correlation between the ages and mean MUP amplitude in the normal and the parkinsonism insertion activity (r =0.766, r =0.659) and the MUR (r =0.89, r = 0.4), respectively. Similarly, there was a correlation between the ages and maximal amplitudes in the normal and parkinsonism groups (r =0.53, r =0.42; r =0.86, r =0.248), respectively, (p<0.001). In the parkinsonism group, there was no significant correlation between the MUs number and the mean MUPs amplitudes in the insertion activity (r= 0.074, p= 0.088) and the MUR (r= 0.226, p=0.762). The spinal cord in the parkinsonism group showed degenerated nerve fibers and apoptosis in the degenerative nerve fibers and in the medium and large motor neurons with Lewy bodies and neurofibrillary tangles. The small ones, however, remained intact. The parkinsonism MUPs, compared to normal ones, have lower threshold and recruit less MUs. The apoptotic medium and large motor neurons with Lewy bodies contribute to the disuse of the relative MUs, while small ones remain intact.

otor unit (MU) is the functional unit of the skeletal muscle that comprises a single motor neuron and muscle fibers which innervates them (1,2). The function of recruited MUs number can make a typical force in the activation of skeletal muscles, but this activity depends on the combination of recruited MUs number (3)(4)(5). In addition, the activity of the upper levels of the central nervous motor system can change in the properties of the MUs even if the spinal cord is intact (6,7). Spinal cord contains several motor neuron pools that relay the messages from the upper brain to the MUs (8,9). The specific central motor processes affect the pools (10,11). The manifestation of certain defective central nervous organs such as substantia nigra in Parkinson's disease (PD) which can interfere to MU activity and the electromyographic (EMG) technique will be able to record MU disorder (12,13). PD is a progressive neurodegenerative disease, characterized by the presence of lewy body, neurofibrillary tangle, and senile plaques (14)(15)(16). PD had long been known as a sole motor system disorder, but later on was discovered to have an effect on the sensation, perception, and emotional functioning (17)(18)(19). Poppele (2001) suggested that apart from the motor system, the sensorimotor such as muscle spindle are also involved in PD (20). Bradykinesia in these patients is accompanied with resting tremor and muscle rigidity that the latter is secondary to the modulation of agonist and antagonist muscular tone (21)(22)(23)(24). The aim of this study was to estimate normal and parkinsonism MUs number in the medial gastrocnemius (MG) muscle in Wistar rats.

Materials and Methods
This experimental study was carried out on to compute MUs number in MG muscle in normal (n=9) and parkinsonism (n=9), adult (154-304 days old) male Wistar rats of the same age groups. The parkinsonism rats were treated with MPTP (Sigma, USA) 10 mg/kg, I.P (25,26). Bradykinesia, tail rigidity, and resting tremor were the predomi-nant sings of PD used to enroll the rats in this study. The rats were anesthetized, using supplemental doses (10 mg/kg, I.P.) of pentobarbital sodium (Sigma, USA) 30 mg/kg, I.P, if necessary. The rats were checked for absence of pinch reflex. Their body temperature was maintained at 37°C by machine control (Harvard Apparatus Limited, USA). At the end of the experiment, the rats were killed through overdosing the anesthetic drug. The left leg's MG muscle was dissected and prepared for direct insertion of the monopolar needle (steel, 0.1-mmdiameter of tip that covered 20-muscle fibers /mm 2 ) of the EMG electrodes into the muscle (13,27).
The EMG signals were recorded after the application of mild to moderate and severe motions of the recording electrodes (equal to 0.05, 0.1mv), considered as insertion activity at rest. Thereafter, two different mechanical involuntary contractions (equal to 0.5, 5mv) were studied on the same muscle. The first two produced voltage, before achieving the threshold as evoked muscle action potential, and the next two voltage evoked maximal amplitude of motor unit potential (MUP) due to motor unit recruitment (MUR).
There were 2-3s intervals between the two tests. The spike potentials were recorded using Power Lab set (ML 866, 4 channels, AD Instruments Co. Australia), which had sweep velocity of 10ms/div, sensitivity of 100 µv/div, and high filtration rate of 1 KHz. The spikes amplitudes (peak-to-peak) were measured, then the means initial (n=10 spikes of each age group) and maximal amplitudes of the MUPs were calculated. Finally the number of units was estimated by dividing these two parameters to each other (28,29). Thereafter a L4-L6 laminectomy was performed; the spinal cord was removed, immersed in 10% formaldehyde solution, and transverse section was stained with congo red for pathological study. Trace A shows normal electromyogram (EMG), in the beginning, motor unit potentials recorded during mild to moderate and maximal involuntary contractions of medial gastrocnemius muscle, in the middle, discharged spike potentials by electrode motions, and the at the end, again evoked maximal spikes. Trace B depicts Parkinsonism EMG, initial evoked spike potentials recorded by electrode motions and recording sporadic with a few high spikes are due to muscle contractions.

Statistical analysis
Student t-test was used to compare amplitude spike potentials in both groups; the p-value <0.05 was considered statistically significant and Pearson correlations wase used to asses the relationship between age and MUPs parameters.

Results
In the last visit, the parkinsonism rats showed typical bradykinesia. If they were impelled to move, they resisted and were agitated at the beginning, they showed clumsiness while moving. Distinctive rigidity in the tail's muscles and resting tremor were clearly visible. Their hair changed from soft to spiky.

MUPs traces
The two samples of the EMG spike potentials traces from the same age group, the normal and parkinsonism groups are shown in fig.1.

Correlation between age and the MU number in normal and parkinsonism
Any increase in age is not exactly associated with increased MU numbers in both groups. The maximal difference of MUs number between the normal and the parkinsonism groups with 181 and 304-day-olds was 82.93% and the minimal difference was 62.85%. These differences were more in the normal group than the parkinsonism group by the insertion activity testing (Table 1, Fig. 2).
Whereas, for the foregoing ages of both groups the maximal difference of 81.33% and the minimal difference of 63.49% were found by the MUR test (     were intact (Fig.4).

Discussion
The present study shows that the normal and parkinsonism MUs of MG muscle have different perception and recruitment which depend on variable power stimuli reception and the next different discharge rate of spike potentials will be evoked. Petit (1990) reported that the MG muscle is a mixed muscle with three different types of fibers innervated with three different types of motor neurons located in spinal cord. They also showed that slow MUs are weaker than the two others (30).